A versatile "pan-spike-in" internal reference genome methodology for quantitative ChIP-seq normalization

用于定量 ChIP-seq 标准化的多功能“pan-spike-in”内部参考基因组方法

基本信息

  • 批准号:
    10323274
  • 负责人:
  • 金额:
    $ 19.06万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2021
  • 资助国家:
    美国
  • 起止时间:
    2021-01-01 至 2023-12-31
  • 项目状态:
    已结题

项目摘要

Project summary: ChIP-seq has enabled the mapping of genomic occupancy of many chromatin factors and epigenetic histone modifications, linked to normal growth and development and to the pathogenesis of many human diseases. ChIP-seq can also measure changes in the occupancy levels of epigenomic marks/factors between different chromatin samples such as control vs drug-treated cells. However, accurate quantitation in ChIP-seq requires effective and reliable sample normalization. ChIP with reference exogenous genome or ChIP- Rx devised by Orlando et al (2014) provides a rigorous normalization approach via addition of crosslinked Drosophila S2 cells (reference exogenous chromatin) with crosslinked human cells on a per-cell basis. Antibody cross-reactivity to the exogenous and experimental test chromatin is critical for the success of this spike-in approach. Exogenous reference species Drosophila, C. glabrata and S. pombe do not share many of the epigenomic/chromatin factors with the experimental human/mouse or S. cerevisiae models. Moreover, lack of “ChIP grade” antibody has led to epitope tagging of chromatin factors. To overcome the antibody cross-reactivity barriers, we hypothesized that a chromatin-binding protein in the exogenous reference genome when fused to the IgG-binding domains of Protein A and G (PAG tag) will cross-react with antibodies raised in rabbit or mouse recognizing epigenomic/chromatin factors or epitope-tags, and thus can serve as an ‘one-for-all or pan spike-in’ for normalization. Based on successfully using a C. glabrata ‘pan spike-in’ exogenous reference for normalization and measurement of chromatin occupancy differences for epigenome-modulating factors in S. cerevisiae, we propose to now create a Drosophila S2R+ ‘pan spike-in’ reference genome for ChIP-Rx in human or mouse cells. We propose to also create S. pombe ‘pan spike-in’ reference genome as a non-pathogenic alternative to C. glabrata, and a ‘pan spike-in’ S. cerevisiae reference for ChIP-Rx in S. pombe, all in Aim 1. We will validate the utility of the created ‘pan-spike-in’ genomes for normalization by measuring the subunit-dependent chromatin occupancy of Set1 H3K4 methyltransferase from yeast to humans using ChIP-Rx (Aim 2). In Aim 3, we propose to diversify the ‘pan-spike-in’ normalization system by testing other immunoglobulin-binding domains to capture goat or chicken IgG. Overall, successful completion of the proposed studies will lead to the creation of valuable reagents for not only epigenomics/chromatin researchers but also to the broader research community, by providing a robust and accurate normalization methodology for ChIP-seq to quantify epigenome differences among cell populations, treatments and genomic states. The studies resulting from using the ‘pan spike-in’ system generated here will yield novel mechanistic insights and also enable accurate quantitation of global and local chromatin modifications that is needed for the discovery and characterization of epigenome regulators and for the drugs targeting them in a variety of human diseases ranging from developmental, metabolic, neurological disorders to cancers.
项目摘要:CHIP-SEQ已经使许多染色质因子的基因组占有率和 表观遗传性组蛋白修饰,与正常生长发育和许多 人类疾病。ChIP-SEQ还可以测量表观基因组标记/因子的占用水平的变化 不同染色质样本之间的差异,例如对照细胞和药物处理细胞。然而,准确的量化在 CHIP-SEQ需要有效和可靠的样本归一化。带有参考外源基因组的芯片或芯片- Orlando等人(2014)设计的RX通过添加交联体提供了严格的标准化方法 果蝇S2细胞(参考外源染色质)在每个细胞的基础上与交叉连接的人类细胞。抗体 对外源和实验测试染色质的交叉反应是这种激增成功的关键 接近。外源参考物种果蝇、光滑果蝇和果蝇不具有许多共同的 表观基因组/染色质因子与实验性的人/鼠或酿酒酵母模型。此外,缺乏 “芯片级”抗体导致了染色质因子的表位标记。克服抗体的交叉反应 障碍,我们假设外源参考基因组中的染色质结合蛋白在融合到 蛋白A和蛋白G的免疫球蛋白结合区(PAG标签)将与兔或小鼠产生的抗体发生交叉反应 识别表观基因组/染色质因子或表位标签,因此可以起到‘一刀切或全盘尖峰’的作用 用于正常化。基于成功利用光肩星天牛的外源参照物进行归一化 和在酿酒酵母中对表观基因组调节因子染色质占有率差异的测量 建议现在为人或小鼠的CHIP-Rx建立一个果蝇S2R+‘PAN尖峰插入’参考基因组 细胞。我们还建议创建Pombe‘PanSpike-in’参考基因组,作为一种非致病替代方案 以及S.pombe中ChIP-Rx的‘PAN尖峰’酿酒酵母参考文献,都在Aim 1中。我们将验证 通过测量依赖于亚单位的染色质,创建的“泛尖峰”基因组用于正常化 使用CHIP-Rx将Set1 H3K4甲基转移酶从酵母转移到人(目标2)。在目标3中,我们建议 通过测试要捕获的其他免疫球蛋白结合结构域来使“泛尖峰”标准化系统多样化 山羊或鸡的免疫球蛋白。总体而言,成功完成拟议的研究将导致创造有价值的 不仅是表观基因组学/染色质研究人员的试剂,而且是更广泛的研究界的试剂, 为CHIP-SEQ提供一种稳健和准确的归一化方法来量化表观基因组差异 在细胞群体、处理和基因组状态之间。通过使用“锅底加压”进行的研究 这里生成的系统将产生新的机械洞察力,并能够准确量化全球和 发现和鉴定表观基因组调节因子所需的局部染色质修饰 针对多种人类疾病的药物,从发育、代谢、神经疾病 从疾病到癌症。

项目成果

期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation.
  • DOI:
    10.1371/journal.pgen.1010376
  • 发表时间:
    2022-08
  • 期刊:
  • 影响因子:
    4.5
  • 作者:
  • 通讯作者:
Targeting DNA Repair and Chromatin Crosstalk in Cancer Therapy.
靶向癌症治疗中的DNA修复和染色质串扰。
  • DOI:
    10.3390/cancers13030381
  • 发表时间:
    2021-01-20
  • 期刊:
  • 影响因子:
    5.2
  • 作者:
    Johnson DP;Chandrasekharan MB;Dutreix M;Bhaskara S
  • 通讯作者:
    Bhaskara S
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Mahesh B Chandrasekharan其他文献

Mahesh B Chandrasekharan的其他文献

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{{ truncateString('Mahesh B Chandrasekharan', 18)}}的其他基金

Structure, function and regulation of the H2B ubiquitin-conjugating complex
H2B 泛素结合复合物的结构、功能和调控
  • 批准号:
    10165311
  • 财政年份:
    2018
  • 资助金额:
    $ 19.06万
  • 项目类别:
Structure, function and regulation of the H2B ubiquitin-conjugating complex
H2B 泛素结合复合物的结构、功能和调控
  • 批准号:
    9923682
  • 财政年份:
    2018
  • 资助金额:
    $ 19.06万
  • 项目类别:
Structure, function and regulation of the H2B ubiquitin-conjugating complex
H2B 泛素结合复合物的结构、功能和调控
  • 批准号:
    10396526
  • 财政年份:
    2018
  • 资助金额:
    $ 19.06万
  • 项目类别:

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使用病毒样颗粒缀合物免疫和高通量选择的合理引导的针对碳水化合物抗原的单克隆抗体的发现平台
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生成最佳非 HRP2 疟疾诊断抗原的特异性抗体
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  • 财政年份:
    2020
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使用进化遥远的海七鳃鳗结构独特的可变淋巴细胞受体抗体询问 B 谱系细胞上的细胞表面抗原
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研究各种天然抗体与食物源性抗原之间的相互作用
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Identifying Kawasaki Disease-Specific Antibodies and Antigens
识别川崎病特异性抗体和抗原
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抗体和抗原之间相互作用的新评分方法
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抗体和抗原之间相互作用的新评分方法
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    2017
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SBIR II 期:针对蛋白质和碳水化合物抗原的抗体的自动化设计方法
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